1. Field of the Invention
This invention relates to nucleic acids, more specifically to nucleic acid analogs, more specifically to nucleic acid analogs that have linking group and base substitutions that make the more stable to degradation, broaden their recognition powers, and improve their ability to form tertiary structures. This spefication makes reference to the patent application entitled xe2x80x9cOligonucleotide Analogs Containing Sulfurxe2x80x9d, Ser. No. 07/202,528 filed: Jun. 6, 1988, issued on Jun. 1, 1993 as U.S. Pat. No. 5,216,141, and Ser. No. 08/068,981, a continuation-in-part of Ser. No. 07/202,528 that is co-pending with this application, and related to Ser. No. 07/594,290, issued as U.S. Pat. No. 5,432,272 on Jul. 11, 1995, and Ser. No. 08/375,132, a continuation-in-part of Ser. No. 07/594,290 that is co-pending with this application. Ser. No. 07/202,528 discloses compositions of matter that are DNA and RNA analogs where the bridging phosphodiester linkages are replaced by dimethylene sulfone groups. The claims that have been allowed in Ser. No. 07/594,290 and that are pending in Ser. No. 08/068,981 refer to compositions where all of the phosphodiester linking groups have been so replaced. The claims in this continuation cover compositions of matter where only some of the linking phosphates are replaced. Since the instant compositions contain both phosphate and dimethylenesulfone linkages, they are referred to as oligonucleotide-sulfone chimeras, or more simply, chimeras. Ser. No. 08/375,132 claims compositions of matter that are oligonucleotide analogs and contain non-standard nucleobases. The compositions claimed in this application includes non-standard nucleobases.
2. Description of the Related Art Background
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are molecules central to biological processes. As oligomers composed of five subunits (adenosine, A, cytidine, C, guanosine, G, uridine, U, and thymidine, T) joined by phosphodiester linkages, naturally occurring nucleic acids possess two notable structural properties.
First, oligonucleotides bind to complementary oligonucleotides (where xe2x80x9ccomplementarity rulesxe2x80x9d are defined such that A in one oligonucleotide strand is paired against either U or T in the other, and G in one oligonucleotide strand is paired against C in the other, with the two strands anti-parallel) to form helical structures. The binding specificity is due to hydrogen bonds formed between bases on one oligonucleotide strand and complementary bases on the other. The thermodynamically stability of the helical structures is in part due to hydrophobic forces between bases stacked on top of each other in the double helical structure.
Second, information is coded in the oligonucleotide by the order of bases in the oligonucleotide strand. This information codes for proteins and nucleic acids necessary for the growth and replication of organisms. Oligonucleotides with define sequence serve as genes, genetic regulatory agents, intracellular messages, especially for the synthesis of proteins, and possibly intercellular messages.
Natural oligonucleotides have widespread value in the laboratory, in diagnostic systems, and as therapeutic agents. However, natural oligonucleotides are degraded in biological environments due to the action of enzymes, particularly deoxyribonucleases (DNases), ribonucleases (RNases), and phosphodiesterases (Plesner, P.; Goodchild, J.; Kalckar, H. M.; Zamecnik, P. C. Proc. Nat. Acad. Sci. 1987 84 1936-1939). This limits their value in many applications.
Many workers have successfully stabilized oligonucleotides to enzymatic degradation by replacing the phosphate linking groups with another. Analogs of oligonucleotides where the phosphate bridging group is replaced by a carboxyl group has also been synthesized (Jones, A. S.; MacCoss, M.; Walker, R. T. Biochem. Biophys. Acta 1973 365 365-377). The analogs slowly hydrolyzed upon standing at neutral pH, and the polymer with adenosine formed an undefined complex with polyuridylic acid. Analogously, carbamate analogs of oligonucleotides have been synthesized (Mungall, W. S.; Kaiser, J. K. J. Org. Chem. 1977 42 703-706). Oligonucleotides have been constructed that contain 1,3 propanediol units between normal phosphate bases (Seela, F.; Kaiser, K. Nucl. Acids Res. 1987 15 3113-3129). These last molecules are not isosteric analogs of DNA, and cannot be prepared in the modified structural forms needed to modulate their binding affinity for natural oligonucleotides (vide infra).
Recently, Miller, T""so, and their coworkers reported the synthesis of compounds that are isosteric analogs of DNA, differing only in that one oxygen of the phosphate group of each subunit is replaced by a methyl group. In all other structural aspects, these molecules are identical to naturally occurring oligonucleotides. These molecules, termed methylphosphonate DNA analogs, or methylphosphonates, lack phosphate-borne negative charges. A patent was recently awarded to the inventors of these molecules (U.S. Pat. No. 4,469,863, Sep. 4, 1984).
However, the methylphosphonates and many similar analogs themselves have several undesirable chemical properties. First, substitution of a methyl group for an oxygen at phosphorus creates a chiral center. Therefore, oligomers composed of methylphosphonate building blocks are again complex mixtures of diastereomers. Further, apparently only one diastereomer of a methylphosphonate-linked dinucleotide can bind to a complementary natural oligonucleotide (Miller, P. S.; Yano, J.; Yano, E.; Carroll, C.; Jayaraman, K.; Ts""o, P.O.P. Biochem. 1979, 18 5134-5143).
A second problem arises from the chemical instability of methylphosphonate diesters and similar analogs. As with triesters of phosphoric acid, diesters of methylphosphonates are readily hydrolyzed in base. Basic conditions are required for the deprotection of bases in the reported procedure for the synthesis of oligomethylphosphonates. Complete deprotection of the bases is critical for full biological activity, as protecting groups block the functional groups that form the hydrogen bonds to complementary oligonucleotides. In the synthesis of natural oligonucleotides, complete deprotection is normally achieved by prolonged exposure of the protected oligonucleotide with base. Phosphodiester groups present in natural oligonucleotides are stable under these conditions. Methylphosphonate diesters are not.
Related applications (Ser. No. 07/202,528, issued, as U.S. Pat. No. 5,216,141, and Ser. No. 08/068,981, copending with this application) discloses oligonucleotide analogs where all linking phosphates in an oligonucleotide are replaced by dimethylene sulfide, sulfoxide, and sulfone units. The claims made in this application differs from previous applications in that they cover compositions of matter that are oligonucleotide analogs, but where only some of the units in the oligomer chain are joined by dimethylenesulfide, sulfoxide, and sulfone units, while others remain phosphodiester linkages or other linker modifications known in the art.
These linkages have advantages over those already known in the art in that they are (in the sulfide and sulfone forms) free of diastereomerism, and are fully stable under both enzymatic and alkaline conditions. Chimeric oligonucleotide analogs have an advantage over the analogs where all phosphodiester linkages are replace by dimethylenesulfone linkers in that they retain some of the negative charges of natural DNA, and therefore retain much of the water-solubility of natural oligonucleotides. Further, fully substituted oligosulfones have a rich conformational versatility through intramolecular interactions that are not obstructed by coulombic repulsion between backbone anionic groups. Fully natural DNA, due to the polyanionic nature of the backbone, exist frequently in an extended structure. Chimera have an intermediate behavior, forming structure in solution more than natural oligonucleotides, but less than fully substituted oligosulfones.